Hsa_circ_0051908 Promotes Hepatocellular Carcinoma Progression by Regulating the Epithelial–Mesenchymal Transition Process

Materials and Methods Hsa_circ_0051908 expression was determined using RT-qPCR. HCC cell proliferation, apoptosis, invasion, and migration were assessed using CCK-8 assay, EdU staining, TUNEL staining, flow cytometry, and transwell assay. The molecular mechanism was analyzed using western blotting. In addition, the role of hsa_circ_0051908 in tumor growth was evaluated in vivo. Results Hsa_circ_0051908 expression was increased in both HCC tissues and cell lines. The proliferation, migration, and invasion of HCC cells were significantly decreased after hsa_circ_0051908 knockdown, while cell apoptosis was notably increased. Furthermore, we found that hsa_circ_0051908 silencing downregulated vimentin and Snail and upregulated E-cadherin. In vivo, hsa_circ_0051908 silencing significantly inhibited the growth of the tumor. Conclusions Our data provide evidence that hsa_circ_0051908 promotes HCC progression partially by mediating the epithelial–mesenchymal transition process, and it may be used for HCC treatment.


Introduction
Hepatocellular carcinoma (HCC) is a highly heterogeneous malignancy that causes death [1].By 2025, more than one million cases of HCC may have been diagnosed globally [2].Although the diagnosis and treatment of HCC have improved, some patients with HCC lose the opportunity for surgery owing to late detection [1][2][3].Moreover, the recurrence rate of HCC metastasis is high [4,5]; therefore, the identification of new biomarkers and therapeutic targets for HCC treatment is warranted.
CircRNAs are a class of RNAs with a relatively stable structure formed by a special loop formation mechanism.They exist widely in eukaryotic cells and regulate tumor development and immune responses [6,7].Several studies indicated that circRNAs regulate the occurrence and malignant progression of HCC [8,9].Wei et al. [10] found that circ-CDYL promotes HCC progression by mediating the noncoding RNA regulatory network and activating the PI3K/AKT and HIF1AN/ Notch2 signaling pathways.Liang et al. [11] found that circβcatenin competitively binds to GSK3β by encoding a short peptide and acts as bait for GSK3β, thus enhancing the stability of β-catenin, promoting its entry into the nucleus, and activating the Wnt/β-catenin pathway.Additionally, a study showed that circUHRF1 can reduce the therapeutic effect of anti-PD-1 in patients with HCC by targeting miR-449c-5p [12].Owing to their unique biological characteristics, stability of closed-loop structure, and differential expression in tissues and body fluids, circRNAs have a wide application prospect in the early diagnosis and treatment of HCC.Furthermore, microarray results showed high expression of hsa_circ_0051908 in liver cancer tissues, indicating that it might play an important role in the development of liver cancer.However, its role in HCC development is still unknown.Therefore, hsa_ circ_0051908 was selected for further evaluation.
Epithelial-mesenchymal transition (EMT) is a biological process in which epithelial cells, which are polarized and organized into layers, lose their characteristics, and acquire mesenchymal phenotypes that are associated with increased motility and invasiveness.EMT promotes cancer cells to detach from the primary tumor, invade surrounding tissues, and metastasize to distant organs [13].The EMT process in HCC involves the activation of several signaling pathways, including the transforming growth factor-β (TGF-β), Wnt/β-catenin, and notch pathways [14].These pathways regulate the expression of EMT-associated transcription factors, such as Snail, Slug, Twist, and ZEB1/ZEB2, which bind to the promoters of genes involved in cell adhesion, cytoskeleton remodeling, and extracellular matrix degradation [15].In addition, the EMT process is associated with poor prognosis and resistance to chemotherapy and immunotherapy in HCC [16].Therefore, targeting EMT and its associated pathways via hsa_circ_0051908 may be a novel approach to treat HCC.
In this study, we explored the expression of hsa_ circ_0051908 in HCC tumors and cell lines and examined the effects of hsa_circ_0051908 silencing on the biological behaviors of HCC cells.Our study findings suggest that hsa_ circ_0051908 may serve as a potential therapeutic target for HCC by regulating the EMT process.

Materials and Methods
2.1.Tissue Specimen Collection.HCC tissues were collected from 70 patients with HCC at Affiliated Cancer Hospital and Institute of Guangzhou Medical University, and experiments using clinical specimens were approved by the ethics committee of Affiliated Cancer Hospital and Institute of Guangzhou Medical University.The inclusion criteria were as follows: (i) HCC diagnosed by radiology, histology, or the International Classification of Disease (ICD) criteria; (ii) participants without any subtype of cancer at the beginning of the study; (iii) age > 18 years.The exclusion criteria were as follows: (i) receiving other antitumor treatments; (ii) lost to followup; and (iii) incomplete clinical data.
2.8.TUNEL Staining.HCC cells were transfected with hsa_ circ_0051908 and NC siRNA.After 48 hr, HCC cells were fixed and the cell membrane was permeated with 4% paraformaldehyde and 0.1% Triton X-100 solution, respectively.The cells were incubated with 50 μL TUNEL solution (Beyotime, C1089), and then nuclei were stained with DAPI, followed by detection using a fluorescence microscope.
2.9.Cell Migration and Invasion Assays.For the migration assay, 2 × 10 4 transfected HCC cells were seeded into the upper transwell chamber (Corning, 3422).The complete medium was added to the 24-well plate (outer chamber).After 48 hr, the fixed cells were stained with 0.4% crystal violet.The migrated cells were observed using a microscope.
For the invasion assay, matrigel (BD, 356234) was polymerized in transwell chambers at 37°C for 60 min.Then, 4 × 10 4 transfected HCC cells were seeded on polymerized matrigel.The subsequent experimental steps were the same as those of the cell migration assay.
2.11.Animal Models.Ten male BALB/c nude mice received a subcutaneous injection of 100 μL HepG2 cells (1 × 10 6 cells).When the tumor volume was approximately 50 mm 3 , the nude mice were randomly divided into two groups (N = 5): NC and siRNA groups.Mice in the NC group were intratumorally injected with 10 nmol NC siRNA and those in the siRNA group were intratumorally injected with 10 nmol hsa_circ_0051908 siRNA.Mice were injected once every 3 days for four times in total.Four weeks after siRNA injections, all mice were euthanized.
2.12.Statistical Analysis.One-way ANOVA and Student's t-test were used to analyze differences among and between the groups, respectively.Data are expressed as mean AE standard deviation (SD).Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad, CA, USA).A P value < 0.05 was considered statistically significant.

Hsa_circ_0051908
Was Upregulated in HCC.Microarray results have shown that hsa_circ_0051908 is highly expressed in liver cancer tissues [17].Here, we found that hsa_circ_0051908 expression was higher in HCC tissues (N = 70) than in adjacent tissues (Figure 1(a)).Moreover, the expression of hsa_ circ_0051908 in normal human hepatocytes (WRL68 cells) and HCC cells (HepG2, Huh7, SK-Hep-1, and SMMC7721) was analyzed using RT-qPCR.The results showed that hsa_ circ_0051908 expression was greatly higher in HCC cells, especially in HepG2 and Huh7 cells, than in WRL68 cells (Figure 1(b)).In addition, the expression of hsa_circ_0051908 was significantly correlated with tumor size, tumor differentiation, TNM stage, and lymph node metastasis (Table 1).
These data suggest that hsa_circ_0051908 might play an important role in the progression of HCC.

Discussion
Clinical research and application of targeted and immunotherapy have enabled some patients with advanced HCC to receive surgical resection during translational therapy.However, delayed diagnosis and ineffective treatment regimens lead to poor prognosis with decreased overall survival in patients with HCC [18,19].Therefore, there is an urgent need to explore diagnostic biomarkers for HCC.In the present study, we found that hsa_circ_0051908 expression was increased in both HCC tissues and cell lines.We further found that hsa_circ_0051908 silencing significantly inhibited tumor growth by regulating the EMT process both in vivo and in vitro.
Increasing evidence indicates that abnormal expression of circRNAs affects the biological characteristics of liver cancer [8,20].For example, circPIP5K1A activates the PI3K-Akt signaling pathway through sponge adsorption of miR-671-5P, thereby promoting the progression of gastric cancer [21]; the circRNA circDLC1 inhibits MMP1-mediated liver cancer progression via interaction with HuR [22]; and exosomal circRNA-100338 promotes HCC metastasis via enhancing invasion and angiogenesis [23].In the current study, hsa_ circ_0051908 was upregulated in HCC tissues, suggesting that it plays an important role in HCC development.To date, the role of hsa_circ_0051908 in liver cancer has not been reported.Here, we showed that the proliferation, migration, and invasion of HCC cells were greatly decreased after hsa_circ_0051908 knockdown, while cell apoptosis was largely increased in vitro.Hsa_circ_0051908 silencing significantly inhibited tumor growth in vivo, suggesting that hsa_ circ_0051908 can promote HCC progression.
The EMT process is crucial for tumor cells to acquire mobility, and it is induced by decreased expression of the epithelial-specific marker E-cadherin and increased expression   Analytical Cellular Pathology of the mesenchymal markers N-cadherin and vimentin [24,25].
Here, upregulated E-cadherin and downregulated vimentin after hsa_circ_0051908 silencing in HCC cells confirmed that hsa_circ_0051908 acts as an oncogene by mediating the EMT process.Snail affects tumor cell metastasis and invasion by participating in the regulation of EMT [26].Snail can bind to the E-box of the E-cadherin gene promoter region to decrease E-cadherin protein expression, thus inducing EMT [26,27].Here, Snail expression was notably decreased after hsa_circ_0051908 silencing, indicating that hsa_circ_0051908 regulated HCC progression via regulating the EMT process.This study has some limitations.In the present study, only the cancer-promoting effect of hsa_circ_0051908 was confirmed in vitro and in vivo.The molecular mechanism of hsa_circ_0051908 in HCC remains unclear.Subsequently, we will aim to elucidate the action mechanism of hsa_ circ_0051908 in HCC progression using bioinformatics analysis and double luciferase and rescue experiments.Our future study will provide a sufficient theoretical basis for the application of hsa_circ_0051908 in liver cancer treatment.
In conclusion, our data indicated that hsa_circ_0051908 is an important regulatory molecule in the development and progression of liver cancer, and the circRNA is expected to be a prognostic indicator and therapeutic target for liver cancer.

TABLE 1 :
The correlation relationship between hsa_circ_0051908 expression and clinical data.